Neuronal degeneration in Alzheimer's disease is linked to aberrant proteolysis, most notably the aberrant breakdown of beta-amyloid precursor protein (BAPP). In this project, pathways available for BAPP proteolysis will be characterized in two human CNS cell lines: the recently introduced human cortical nerve cell line "HCN-1A", and the astrocytic cell line U-373 MG. Four specific aims are proposed: 1. To characterize neuronal versus astrocytic BAPP protease activities in vitro -- assessing cleavage sites, compartmentalization, inhibitor sensitivity, and dependence on differentiation. 2. To identify catabolites, and their cleavage sites, produced by intact cells from normal and mutated forms of BAPP. 3. To discover factors that alter the prevalence of specific proteolytic pathways for BAPP turnover. 4. To test the role of altered BAPP proteolytic pathways, and the consequences of their inhibition, in neurodegenerative cascades. These aims rely critically on technical collaborations with projects 1-4, including key methods for in vitro assays of BAPP protease activities, generation of essential antisera, cleavage site analysis of immunoprecipitated catabolites, and the preparation of transfected cells. Advanced microscopic methods to be used include confocal fluorescence imaging, VEC-DIC microscopy, and whole-mount transmission electron microscopy. Confocal fluorescence imaging also will be used, as part of AIM 4 and in collaboration with projects 2 and 4, to examine brain sections from human patients and from transgenic animals. Completion of this project will give significant new insight into the regulatory cell biology of alternative proteolytic pathways for BAPP breakdown in cells from the human CNS, including pathways of direct relevance to Alzheimer's pathogenesis and treatment. The project will identify alternative BAPP proteases in human brain cells, elucidate factors that alter protease activities, and help verify whether specific protease inhibitors will be of use in preventing BAPP-associated neurodegeneration.